Nucleic acid sequencing system and method

ABSTRACT

A technique for sequencing nucleic acids in an automated or semi-automated manner is disclosed. Sample arrays of a multitude of nucleic acid sites are processed in multiple cycles to add nucleotides to the material to be sequenced, detect the nucleotides added to sites, and to de-block the added nucleotides of blocking agents and tags used to identify the last added nucleotide. Multiple parameters of the system are monitored to enable diagnosis and correction of problems as they occur during sequencing of the samples. Quality control routines are run during sequencing to determine quality of samples, and quality of the data collected.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/878,687, entitled “Nucleic Acid Sequencing System and Method,” filedSep. 9, 2010, which is herein incorporated in its entirety by reference,and which is a continuation of U.S. patent application Ser. No.12/020,721, entitled “Nucleic Acid Sequencing System and Method,” filedJan. 28, 2008, and issued as U.S. Pat. No. 7,835,871 on Nov. 16, 2010,which is herein incorporated in its entirety by reference, and whichclaims priority of U.S. Provisional Patent Application No. 60/897,646,entitled “Image Data Efficient Genetic Sequencing Method and System,”filed Jan. 26, 2007, which is herein incorporated in its entirety byreference, and of U.S. Provisional Patent Application No. 60/897,647,entitled “Nucleic Acid Sequencing System and Method,” filed Jan. 26,2007, which is herein incorporated in its entirety by reference.

BACKGROUND

The present invention relates generally to the field of geneticsequencing. More particularly, the invention relates to improvedtechniques for permitting automated sequencing of genetic materials byuse of arrays of genetic fragments.

Genetic sequencing has become an increasingly important area of geneticresearch, promising future uses in diagnostic and other applications. Ingeneral, genetic sequencing consists of determining the order ofnucleotides for a nucleic acid such as a fragment of RNA or DNA.Relatively short sequences are typically analyzed, and the resultingsequence information may be used in various bioinformatics methods toalign fragments against a reference sequence or to logically fitfragments together so as to reliably determine the sequence of much moreextensive lengths of genetic material from which the fragments werederived. Automated, computer-based examination of characteristicfragments have been developed, and have been used more recently ingenome mapping, analysis of genetic variation between individuals,identification of genes and their function, and so forth. However,existing techniques are highly time-intensive, and resulting genomicinformation is accordingly extremely costly.

A number of alternative sequencing techniques are presently underinvestigation and development. These include the use of microarrays ofgenetic material that can be manipulated so as to permit paralleldetection of the ordering of nucleotides in a multitude of fragments ofgenetic material. The arrays typically include many sites formed ordisposed on a substrate. Additional materials, typically singlenucleotides or strands of nucleotides (oligonucleotides) are introducedand permitted or encouraged to bind to the template of genetic materialto be sequenced, thereby selectively marking the template in a sequencedependent manner. Sequence information may then be gathered by imagingthe sites. In certain current techniques, for example, each nucleotidetype is tagged with a fluorescent tag or dye that permits analysis ofthe nucleotide attached at a particular site to be determined byanalysis of image data. Although such techniques show promise forsignificantly improving throughput and reducing the cost of sequencing,further progress in the speed and reliability of the analytical stepsinvolved in sequencing is desirable.

BRIEF DESCRIPTION

The present invention provides significant improvements in the field ofnucleic acid sequencing, especially with regard to instrumentation andanalysis methods. The techniques may be used for any desired sequencing,and will typically be most useful in sequencing of DNA and RNA(including cDNA). The techniques are based upon analysis of nucleotidesequences in samples supported on a substrate, and typically containinga multitude of individual sites such as in a nucleic acid array.Moreover, the techniques may be used with a variety of sequencingapproaches or technologies, including techniques often referred to assequencing-by-synthesis (SBS), sequencing-by-ligation, pyrosequencingand so forth. The present techniques have been found or are believed toprovide for more highly automated or higher quality sequencing,permitting higher throughput and ultimately reduced sequence costs.

Accordingly, the invention provides a method for sequencing a pluralityof nucleic acids which can include the steps of (a) beginning a cycle ofa sequencing procedure for an array having a plurality of nucleic acidsvia a system capable of determining nucleotide sequence of the array;(b) evaluating a parameter of the system; (c) altering the sequencingprocedure for the array based on the parameter; and (d) performinganother cycle of the sequencing procedure for the array.

The invention further provides a method for sequencing a plurality ofnucleic acids which includes the steps of (a) performing an automatednucleic acid sequencing operation; (b) generating data based upon theoperation; and (c) evaluating a quality of the sample based upon thedata.

Also provided is a method for sequencing a plurality of nucleic acids,including the steps of (a) performing a cycle of a sequencing procedurefor an array having a plurality of nucleic acids via a system capable ofdetermining nucleotide sequence of the array; (b) detecting a pluralityof signals indicative of nucleotides present at sites of the array; (c)evaluating the signals to determine quality of the array; and (d)altering the sequencing procedure for the array based on the quality.

A method for sequencing a plurality of nucleic acids can include stepsof (a) introducing a process fluid to an array or nucleic acids in asystem performing a nucleic acid sequencing procedure for the array; and(b) performing via the system at least one cycle of the sequencingprocedure for the array; wherein the process fluid is heated or cooledprior to introduction to the array.

A method for sequencing a plurality of nucleic acids can include thesteps of (a) performing a cycle of a sequencing procedure for an arraycomprising a plurality of nucleic acids via a system capable ofdetermining nucleotide sequence of the array; (b) detecting a pluralityof signals indicative of nucleotides present at sites of the array; and(c) repeating steps (a) and (b); wherein scheduling of steps (a) and (b)is temporally decoupled.

A method for sequencing a plurality of nucleic acids can include thesteps of (a) performing a cycle of a sequencing procedure for an arrayhaving a plurality of nucleic acids via a system capable of determiningnucleotide sequence of the array; (b) imaging the array to generateimage data; (c) deriving sequence data from the image data, the sequencedata indicative of nucleotide species present at a position in thesequence of a nucleic acid of the array; (d) repeating steps (a), (b)and (c); and (e) retaining the sequence data and deleting at least aportion of the image data from which the sequence data was derived priorto completion of the sequencing procedure on the array.

The invention also provides a system for sequencing a plurality ofnucleic acids. The system can include a plurality of processing stationsconfigured to add tagged nucleotides to sites of an array; and aplurality of detecting stations interspersed with the processingstations for detecting nucleic acid sequences of the sites andgenerating data representative thereof.

Also provided is a system for sequencing a plurality of nucleic acidsincluding a fluidics handling system for facilitating assay reactionprotocols; an imaging system for acquiring sequencing data; diagnosticcomponents configured to measure system parameters during operation ofthe sequencing system; quality evaluation circuitry configured to assessa quality of the sequencing system based upon a multiple step analysis;and control circuitry configured to alter operating conditions of thesequencing system based upon data collected by the diagnostic componentsor the quality evaluation circuitry.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a diagrammatical overview of a sequencing system incorporatingaspects of the present technique;

FIG. 2 is a diagrammatical overview of a multi-station sequencing systemimplementing aspects of the present technique;

FIG. 3 is a diagrammatical overview of an exemplary imaging system thatmay be used in conjunction with the system of FIG. 1 or 2 for detectionof sequences at individual sites in an array;

FIG. 4 is a diagrammatical representation of sequencing in the systemsof the preceding figures in accordance with an SBS technique, as oneexample of the sequencing approach that may be used in the systems;

FIG. 5 is a flow chart illustrating exemplary logic for control of thesequencing and sample quality in accordance with aspects of the presenttechnique;

FIG. 6 is a flow chart illustrating exemplary logic for an initialsequencing cycle quality control approach in accordance with aspects ofthe present technique, such as to determine quality of the sample to betested;

FIG. 7 is a flow chart illustrating exemplary logic for a control ofquality of base or nucleotide addition in accordance with the presenttechnique; and

FIG. 8 is a flow chart illustrating exemplary logic for de-blockingquality control in accordance with aspects of the present technique.

DETAILED DESCRIPTION

Turning now to the drawings, referring first to FIG. 1, a diagrammaticalrepresentation of a sequencing system 10 is illustrated as including asequencer 12 designed to determine sequences of genetic material of asample 14. The sequencer may function in a variety of manners, and basedupon a variety of techniques, including sequencing by primer extensionusing labeled nucleotides, as in a presently contemplated embodiment, aswell as other sequencing techniques such as sequencing by ligation orpyrosequencing. In general, and as described in greater detail below,the sequencer 12 progressively moves samples through reaction cycles andimaging cycles to progressively build oligonucleotides by bindingnucleotides to templates at individual sites on the sample. In a typicalarrangement, the sample will be prepared by a sample preparation system16. This process may include amplification of fragments of DNA or RNA ona support to create a multitude of sites of DNA or RNA fragments thesequence of which are determined by the sequencing process. Exemplarymethods for producing sites of amplified nucleic acids suitable forsequencing include, but are not limited to, rolling circle amplification(RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998)), bridge PCR (Adamsand Kron, Method for Performing Amplification of Nucleic Acid with TwoPrimers Bound to a Single Solid Support, Mosaic Technologies, Inc.(Winter Hill, Mass.); Whitehead Institute for Biomedical Research,Cambridge, Mass., (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000);Pemov et al., Nucl. Acids Res. 33:e11 (2005); or U.S. Pat. No.5,641,658), polony generation (Mitra et al., Proc. Natl. Acad. Sci. USA100:5926-5931 (2003); Mitra et al., Anal. Biochem. 320:55-65 (2003)), orclonal amplification on beads using emulsions (Dressman et al., Proc.Natl. Acad. Sci. USA 100:8817-8822 (2003)) or ligation to bead-basedadapter libraries (Brenner et al., Nat. Biotechnol. 18:630-634 (2000);Brenner et al., Proc. Natl. Acad. Sci. USA 97:1665-1670 (2000));Reinartz, et al., Brief Funct. Genomic Proteomic 1:95-104 (2002)). Thesample preparation system 16 will typically dispose the sample, whichmay be in the form of an array of sites, in a sample container forprocessing and imaging.

The sequencer 12 includes a fluidics control/delivery system 18 and adetection system 20. The fluidics control/delivery system 18 willreceive a plurality of process fluids as indicated generally byreference numeral 22, for circulation through the sample containers ofthe samples in process, designated generally by reference numeral 24. Aswill be appreciated by those skilled in the art, the process fluids willvary depending upon the particular stage of sequencing. For example, inSBS using labeled nucleotides, the process fluids introduced to thesample will include a polymerase and tagged nucleotides of the fourcommon DNA types, each nucleotide having a unique fluorescent tag and ablocking agent linked to it. The fluorescent tag allows the detectionsystem 20 to detect which nucleotides were last added to probeshybridized to template nucleic acids at individual sites in the array,and the blocking agent prevents addition of more than one nucleotide percycle at each site. In other processes, such as sequencing by ligation,the process fluids at this stage will include query probes with uniquefluorescent tags attached thereto. Similarly, the query probes will bindto the templates at each site in a configuration that allows ligation ofthe query probes to an anchor primer and may be detected by thedetection system 20 for sequencing of the templates at each site.

At other phases of the sequencing cycles, the process fluids 22 willinclude other fluids and reagents such as reagents for removingextension blocks from nucleotides, cleaving nucleotide linkers, or forremoving bases from ligated oligonucleotides to release a newlyextendable probe terminus. For example, once reactions have taken placeat individual sites in the array of the samples, the initial processfluid containing the tagged nucleotides will be washed from the samplein one or more flushing operations. The sample may then undergodetection, such as by the optical imaging at the detection system 20.Subsequently, reagents will be added by the fluidics control/deliverysystem 18 to de-block the last added nucleotide and remove thefluorescent tag from each. The fluidics control/delivery system 18 willtypically then again wash the sample, which is then prepared for asubsequent cycle of sequencing. Exemplary fluidic and detectionconfigurations that can be used in the methods and devices set forthherein are described in WO 07/123744. In general, such sequencing maycontinue until the quality of data derived from sequencing degrades dueto cumulative loss of yield or until a predetermined number of cycleshave been completed, as described in greater detail below.

The quality of samples 24 in process as well as the quality of the dataderived by the system, and the various parameters used for processingthe samples is controlled by a quality/process control system 26. Thequality/process control system 26 will typically include one or moreprogrammed processors, or general purpose or application-specificcomputers which communicate with sensors and other processing systemswithin the fluidics control/delivery system 18 and the detection system20. A number of process parameters, discussed in further detail below,may be used for sophisticated quality and process control, for example,as part of a feedback loop that can change instrument operationparameters during the course of a sequencing run.

The sequencer 12 also communicates with a system control/operatorinterface 28 and ultimately with a post-processing system 30. Hereagain, the system control/operator interface 28 will typically include ageneral purpose or application-specific computer designed to monitorprocess parameters, acquired data, system settings, and so forth. Theoperator interface may be generated by a program executed locally or byprograms executed within the sequencer 12. In general, these may providevisual indications of the health of the systems or subsystems of thesequencer, the quality of the data acquired, and so forth. The systemcontrol/operator interface 28 may also permit human operators tointerface with the system to regulate operation, initiate and interruptsequencing, and any other interactions that may be desired with thesystem hardware or software. For instance, the system control/operatorinterface 28 may automatically undertake and/or modify steps to beperformed in a sequencing procedure, without input from a humanoperator. Alternatively or additionally, the system control/operatorinterface 28 may generate recommendations regarding steps to beperformed in a sequencing procedure and display these recommendations tothe human operator. This mode would, of course, allow for input from thehuman operator before undertaking and/or modifying steps in thesequencing procedure. In addition, the system control/operator interface28 may provide an option to the human operator allowing the humanoperator to select certain steps in a sequencing procedure to beautomatically performed by the sequencer 12 while requiring input fromthe human operator before undertaking and/or modifying other steps. Inany event, allowing both automated and operator interactive modes mayprovide increased flexibility in performing the sequencing procedure. Inaddition, the combination of automation and human-controlled interactionmay further allow for a system capable of creating and modifying newsequencing procedures and algorithms through adaptive machine learningbased on the inputs gathered from human operators.

The post-processing system 30 will typically also include one or moreprogrammed computers that receive detected information, which may be inthe form of pixilated image data and derive sequence data from the imagedata. The post-processing system 30 may include image recognitionalgorithms which distinguish between colors of dyes attached tonucleotides that bind at individual sites as sequencing progresses(e.g., by analysis of the image data encoding specific colors orintensities), and logs the sequence of the nucleotides at the individualsite locations. Progressively, then, the post-processing system 30 willbuild sequence lists for the individual sites of the sample array whichcan be further processed to establish genetic information for extendedlengths of material by various bioinformatics algorithms.

The sequencing system 10 may be configured to handle individual samplesor may be designed for higher throughput in a manner generallyrepresented in FIG. 2. FIG. 2 illustrates a multi-station sequencer 32in which multiple stations are provided for the delivery of reagents andother fluids, and for detection of progressively building sequences ofnucleotides. In the illustrated embodiment, the sequencer 32 may includea series of stations disposed in a plane, such as on a table, or inmultiple planes. To allow samples to be inserted into the sequencer, aninsertion/retrieval station 34 will typically be provided. This stationwill be physically configured to allow a human operator or robot toinsert a sample into the device and lodge the sample in a process flowfor sequencing operations to be automatically performed at the variousadditional stations. From the insertion/retrieval station 34, amechanical conveying system (not illustrated) will serve to move thesamples 24 and process between the other stations.

In the embodiment illustrated in FIG. 2, the additional stations willinclude fluidic stations 36, detection stations 38, and de-blockingstations 40, although other stations may be included or interspersedwith these stations depending upon the process and sequence of stepsdesired. For example, fluidic stations 36 will serve to introducereagents and other process fluids to the samples 24, such as to allowfor binding of individual nucleotides as sequencing progresses. Thefluidic stations 36 may also allow for washing or flushing reagents fromthe samples. Alternatively or additionally, the stage supporting thesample can be configured to allow removal of liquids, including reagentspresent in the liquids, from samples independent of their location inthe system. For example, the stage can include valve actuated vacuumlines that can be activated for removal of liquids from the sample whenthe sample is at any station or even when the sample is betweenstations. A useful vacuum system is described, for example, in pendingU.S. patent application Ser. No. 11/521,574, which is incorporatedherein by reference.

The detection stations 38 may include any desired detection circuitry,such as optical, electrical, or other equipment designed to detect theparticular nucleotides added at individual sites of the sample as thesequencing progresses. An exemplary optical system for such detection isdescribed below with reference to FIG. 3. The de-blocking station 40 maybe employed for delivering reagents used to remove protective moleculesthat prevent binding of more than one nucleotide at a time, particularlyin SBS systems. The de-blocking station 40 may also be used to cleavefluorescent dyes and similar molecules from the nucleotides oroligonucleotides as sequencing progresses.

In general, the samples 24 may progress through the sequencer 32 in aprogressive flow direction as indicated generally by arrow 42. This maycorrespond to a normal flow of the sample through the sequencer.However, the samples may retrogress in the stations as indicatedgenerally by reference numeral 44. Such retrogression may be desired topermit re-imaging of the samples, reintroduction of reagents,re-flushing, or generally any repetitive operation that can be performedby a preceding station. It should also be noted that the progression ofsamples in the system, as also in the system of FIG. 1, may be decoupledin a temporal sense. That is, not all samples need to progress throughthe stations for the same number of cycles nor do all samples need toenter and exit a multi-cycle process in the same cycle.

Samples may be removed from processing, reprocessed, and scheduling ofsuch processing may be altered in real time, particularly where thefluidics control system 18 or the quality/process control system 26detect that one or more operations were not performed in an optimal ordesired manner. In embodiments wherein a sample is removed from theprocess or experiences a pause in processing that is of a substantialduration, the sample can be placed in a storage state. Placing thesample in a storage state can include altering the environment of thesample or the composition of the sample to stabilize biomoleculereagents, biopolymers or other components of the sample. Exemplarymethods for altering the sample environment include, but are not limitedto, reducing temperature to stabilize sample constituents, addition ofan inert gas to reduce oxidation of sample constituents, and removingfrom a light source to reduce photobleaching or photodegradation ofsample constituents. Exemplary methods of altering sample compositioninclude, without limitation, adding stabilizing solvents such asantioxidants, glycerol and the like, altering pH to a level thatstabilizes enzymes, or removing constituents that degrade or alter otherconstituents. In addition, certain steps in the sequencing procedure maybe performed before removing the sample from processing. For instance,if it is determined that the sample should be removed from processing,the sample may be directed to the fluidics control/delivery system 18 sothat the sample may be washed before storage. Again, these steps may betaken to ensure that no information from the sample is lost.

Moreover, sequencing operations may be interrupted by the sequencer 12at any time upon the occurrence of certain predetermined events. Theseevents may include, without limitation, unacceptable environmentalfactors such as undesirable temperature, humidity, vibrations or straylight; inadequate reagent delivery or hybridization; unacceptablechanges in sample temperature; unacceptable sample sitenumber/quality/distribution; decayed signal-to-noise ratio; insufficientimage data; and so forth. It should be noted that the occurrence of suchevents need not require interruption of sequencing operations. Rather,such events may be factors weighed by the quality/process control system26 in determining whether sequencing operations should continue. Forexample, if an image of a particular cycle is analyzed in real time andshows a low signal for that channel, the image can be re-exposed using alonger exposure time, or have a particular chemical treatment repeated.If the image shows a bubble in a flow cell, the instrument canautomatically flush more reagent to remove the bubble, then re-recordthe image. If the image shows zero signal for a particular channel inone cycle due to a fluidics problem, the instrument can automaticallyhalt scanning and reagent delivery for that particular channel, thussaving on analysis time and reagent consumption.

Although the system has been exemplified above with regard to a systemin which a sample interfaces with different stations by physicalmovement of the sample, it will be understood that the principles setforth herein are also applicable to a system in which the stepsoccurring at each station are achieved by other means not requiringmovement of the sample. For example, reagents present at the stationscan be delivered to a sample by means of a fluidic system connected toreservoirs containing the various reagents. Similarly, an optics systemcan be configured to detect a sample that is in fluid communication withone or more reagent stations. Thus, detection steps can be carried outbefore, during or after delivery of any particular reagent describedherein. Accordingly, samples can be effectively removed from processingby discontinuing one or more processing steps, be it fluid delivery oroptical detection, without necessarily physically removing the samplefrom its location in the device.

As in the system of FIG. 1, the various stations are coupled to thefluidics control system 18 and to the quality/process control system 26to permit control of these operations, as well as control of quality ofboth the samples and of the operations performed at the variousprocessing stations. Moreover, as in the system of FIG. 1, the variousstations of the sequencer are linked to a system control/operatorinterface 28, and data collected is ultimately forwarded to apost-processing system 30 where sequence data is derived from thedetected data, typically image data generated by the detection stations38.

A system of the invention can be used to continuously sequence nucleicacids in a plurality of different samples. Systems of the invention canbe configured to include an arrangement of samples and an arrangement ofstations for carrying out sequencing steps. The samples in thearrangement of samples can be placed in a fixed order and at fixedintervals relative to each other. For example, an arrangement of nucleicacid arrays can be placed along the outer edge of a circular table.Similarly, the stations can be placed in a fixed order and at fixedintervals relative to each other. For example, the stations can beplaced in a circular arrangement having a perimeter that corresponds tothe layout for the arrangement of sample arrays. Each of the stationscan be configured to carry out a different manipulation in a sequencingprotocol. The two arrangements (i.e. sample arrays and stations) can bemoved relative to each other such that the stations carry out desiredsteps of a reaction scheme at each reaction site. The relative locationsof the stations and the schedule for the relative movement can correlatewith the order and duration of reaction steps in the sequencing reactionscheme such that once a sample array has completed a cycle ofinteracting with the full set of stations, then a single sequencingreaction cycle is complete. For example, primers that are hybridized tonucleic acid targets on an array can each be extended by addition of asingle nucleotide, detected and de-blocked if the order of the stations,spacing between the stations, and rate of passage for the arraycorresponds to the order of reagent delivery and reaction time for acomplete sequencing reaction cycle.

In accordance with the configuration set forth above, and described infurther detail below, each lap (or full revolution in embodiments wherea circular table is used) completed by an individual sample array cancorrespond to determination of a single nucleotide for each of thetarget nucleic acids on the array (i.e. including the steps ofincorporation, imaging, cleavage and de-blocking carried out in eachcycle of a sequencing run). Furthermore, several sample arrays presentin the system (for example, on the circular table) concurrently movealong similar, repeated laps through the system, thereby resulting incontinuous sequencing by the system. Using a system or method of theinvention, reagents can be actively delivered or removed from a firstsample array in accordance with a first reaction step of a sequencingcycle while incubation, or some other reaction step in the cycle, occursfor a second sample array. Thus, a set of stations can be configured ina spatial and temporal relationship with an arrangement of sample arrayssuch that reactions occur at multiple sample arrays concurrently even asthe sample arrays are subjected to different steps of the sequencingcycle at any given time, thereby allowing continuous and simultaneoussequencing to be performed. The advantages of such a circular system areapparent when the chemistry and imaging times are disproportionate. Forsmall flow cells that only take a short time to scan, it is advantageousto have a number of flow cells running in parallel in order to optimizethe time the instrument spends acquiring data. When the imaging time andchemistry time are equal, a system that is sequencing a sample on asingle flow cell spends half the time performing a chemistry cyclerather than an imaging cycle, and therefore a system that can processtwo flow cells could have one on the chemistry cycle and one on theimaging cycle. When the imaging time is ten fold less than the chemistrytime, the system can have ten flow cells at various stages of thechemistry process whilst continually acquiring data.

Embodiments of the invention provide a system that is configured toallow replacement of a first sample array with a second sample arraywhile the system continuously sequences nucleic acids of a third samplearray. Thus, a first sample array can be individually added or removedfrom the system without interrupting sequencing reactions occurring atanother sample array, thereby providing the advantage of continuoussequencing for the set of sample arrays. A further advantage is thatsequencing runs of different lengths can be performed continuously andsimultaneously in the system because individual sample arrays cancomplete a different number of laps through the system and the samplearrays can be removed or added to the system in an independent fashionsuch that reactions occurring at other sites are not perturbed.

FIG. 3 illustrates an exemplary detection station 38 designed to detectnucleotides added at sites of an array in accordance with a presentlycontemplated optical system. As set forth above, a sample can be movedto two or more stations of the device that are located in physicallydifferent locations or alternatively one or more steps can be carriedout on a sample that is in communication with the one or more stationswithout necessarily being moved to different locations. Accordingly, thedescription herein with regard to particular stations is understood torelate to stations in a variety of configurations whether or not thesample moves between stations, the stations move to the sample, or thestations and sample are static with respect to each other. In theembodiment illustrated in FIG. 3, one or more light sources 46 providelight beams that are directed to conditioning optics 48. The lightsources 46 may include one or more lasers, with multiple laserstypically being used for detecting dyes that fluoresce at differentcorresponding wavelengths. The light sources may direct beams to theconditioning optics 48 for filtering and shaping of the beams in theconditioning optics. For example, in a presently contemplatedembodiment, the conditioning optics 48 combine beams from multiplelasers and generate a generally linear beam of radiation that isconveyed to focusing optics 50. The laser modules can additionallyinclude a measuring component that records the power of each laser. Themeasurement of power may be used as a feedback mechanism to control thelength of time an image is recorded in order to obtain a uniformexposure energy, and therefore signal, for each image. If the measuringcomponent detects a failure of the laser module, then the instrument canflush the sample with a “holding buffer” to preserve the sample untilthe error in the laser can be corrected.

The sample 24 is positioned on a sample positioning system 52 that mayappropriately position the sample in three dimensions, and may displacethe sample for progressive imaging of sites on the sample array. In apresently contemplated embodiment, the focusing optics 50 confocallydirect radiation to one or more surfaces of the array at whichindividual sites are located that are to be sequenced. Depending uponthe wavelengths of light in the focused beam, a retrobeam of radiationis returned from the sample due to fluorescence of dyes bound to thenucleotides at each site.

The retrobeam is then returned through retrobeam optics 54 which mayfilter the beam, such as to separate different wavelengths in the beam,and direct these separated beams to one or more cameras 56. The cameras56 may be based upon any suitable technology, such as including chargecoupled devices that generate pixilated image data based upon photonsimpacting locations in the devices. The cameras generate image data thatis then forwarded to image processing circuitry 58. In general, theprocessing circuitry 58 may perform various operations, such asanalog-to-digital conversion, scaling, filtering, and association of thedata in multiple frames to appropriately and accurately image multiplesites at specific locations on the sample. The image processingcircuitry 58 may store the image data, and will ultimately forward theimage data to the post-processing system 30 where sequence data can bederived from the image data. Particularly useful detection devices thatcan be used at a detection station include, for example, those describedin US 2007/0114362 (U.S. patent application Ser. No. 11/286,309) and WO07/123744, each of which is incorporated herein by reference.

FIG. 4 illustrates a typical reaction cycle in a sequencing by synthesistechnique for oligonucleotides that may benefit from the nucleotiderecapture and recycling technique of the present invention. In general,the synthesis operation summarized in FIG. 4 may be performed on asample 24 comprising a support 60 on which a multitude of sites 62 and64 are formed. In the preparation of each sample 24, many such sites maybe formed, each with unique fragments of genetic material as indicatedgenerally by reference numeral 66. These fragments may constitutetemplates of DNA or RNA to be sequenced. The fragments can be isolatedfrom a biological source using methods known in the art. In embodimentsutilizing amplification methods, the fragments can be amplicons of a DNAor RNA isolated from a biological source. Each template comprises anumber of mers or bases 68 which will uniquely bind to a complimentarynucleotide (or analog thereof) during the synthesis process. Thesequencing process begins with binding of an anchor primer 70 to each ofthe templates. This anchor primer includes complementary bases 72 thatbind with those of a portion of a template sequence. The remainingportion of the template, designated generally by reference numeral 74,constitutes that portion to be sequenced. The length 76 of the portionto be sequenced may vary, with presently contemplated embodimentsextending from 25 to 40 bases or even as many as 50, 75, or 100 bases.

As sequencing progresses, the introduced processed stream will includeall four common DNA nucleotides, one of which will add to the primer ata position that is opposite the next available base in the template, asindicated by reference numeral 78. The added nucleotide will include abase 80 that is complementary to the template as well as a fluorescenttag 82 and a blocking molecule 84. As will be noted by those skilled inthe art, as used herein, the term “nucleotides” in the illustratedprocesses will typically include units from which DNA molecules areconstructed. Although any nucleotides or oligonucleotides may berecaptured and recycled in accordance with the present technique, inmany practical applications these will includedeoxynucleotide-triphosphates (dNTP), each carrying a single nitrogenousbase (adenine, guanine, cytosine or thymine). The complementarynucleotide is added to the primer due to the activity of a polymerase,as indicated generally by reference numeral 86. Other nucleotides thanthe specific one binding to the template will also be present in theprocess fluid, as indicated generally by reference numerals 88, 90, and92 in FIG. 4. Nucleotides not binding to the templates will subsequentlybe washed from the sample in a flushing operation, exiting in theeffluent stream to be recaptured and recycled as described above.

The sequencing system utilized of the type described above for analysisof oligonucleotide sequences may be automated and regulated in a numberof ways. The present technique provides for automatic detection of anumber of parameters of such systems and control of the sequencingprocess based upon such parameters. In general, the performance andquality control implemented by the present invention may allow fornormal sequencing operations on one or many sample arrays, which may bealtered based upon detected issues with performance or quality of thesample array, performance of the fluidics control/delivery system,performance of the detection system, or any subcomponent or subsystem ofthese. When exceptions or anomalies in quality or performance aredetected, as described in greater detail below, remedial measures may betaken to correct the system performance, re-sequence or re-run certainsequencing cycle steps, such as nucleotide addition, imaging,de-blocking and so forth, or even interrupt sequencing altogether.Because the sequencing will represent an investment in terms of time andmaterials, the remedial measures may be adapted to continue sequencingif at all possible, while taking steps to guard against pursuing asynthesis procedure that is destined to fail or at least destined toproduce results that are not of sufficient value to warrant the time andmaterials spent. Thus, the remedial measures improve the likelihood thatreliable sequencing data will be obtained.

FIG. 5 represents exemplary logic for carrying out and controlling asequencing operation in accordance with this approach. The sequencingoperation, denoted generally by reference numeral 94, begins withloading a sample array in the sequencing system, as indicated at step96. As noted above, a number of different approaches may be employed, asmay various configurations of arrays. In a presently contemplatedembodiment, for example, arrays of a multitude of genetically differentsites are employed, with each site being populated by a multitude of thesame oligonucleotide, template, or fragment to be sequenced. The arraymay be loaded in a sample container and coupled to the fluidicscontrol/delivery system such that reagents and other process fluids canbe introduced to the sample and routed through the sample container forreactions (e.g., base addition and de-blocking), flushing, and so forth.

An array used in the invention can be any population of differentreaction sites that are present at one or more substrates such thatdifferent reaction sites can be differentiated from each other accordingto their relative location. Typically, a single species of biopolymer,such as a nucleic acid, is attached at each individual reaction site.However, multiple copies of a particular species of biopolymer can beattached at a particular reaction site. The array taken as a whole willtypically include a plurality of different biopolymers attached at aplurality of different sites. The reaction sites can be located atdifferent addressable locations on the same substrate. Alternatively, anarray can include separate substrates, such as beads, each bearing adifferent reaction sites.

At step 98 in FIG. 5, then, bases or nucleotides (or oligonucleotides inthe case of processes such as sequencing-by-ligation orsequencing-by-hybridization) are added to the sites in the array inaccordance with the particular sequencing approach adopted. Otherbiomolecule reagents used at this step can also be delivered including,for example, enzymes such as polymerase or ligase. For example, in SBS,polymerase and the four common nucleotide types, each including blockingagents and unique fluorescent dyes are introduced to the sample arrayand are allowed to react with the oligonucleotide templates at eachsite. Step 98 would also include, then, flushing the samples of thepolymerase and nucleotides once sufficient time has elapsed for thedesired reactions. At step 100, the sites and the most recently attachednucleotides are detected. As noted above, this detection may beperformed in a variety of manners, with optical detection being favoredin a presently contemplated embodiment. As also described above, thedetection step can include progressively scanning the sites on the arrayto produce image data which is processed to identify individual sitesand, ultimately, the identity of the most recently attached nucleotidesat each site.

At step 102, the logic determines whether the current cycle is theinitial sequencing cycle. As noted above, sequencing may include anumber of similar cycles of base addition, detection, and de-blocking,with from 25 to 40 or even more such cycles being presentlycontemplated. If the current cycle, then, is the initial cycle, aninitial cycle quality control routine is performed as indicated at step104. This routine may be configured to determine one or more qualitiesof the array as described in greater detail below with reference to FIG.6. It should be noted that the initial cycle quality control routine maycause corrections to be made in the sequencing system, or may cause analteration in the manner in which the individual sample array ishandled. That is, certain steps may be re-performed, or system changesmay be made based upon the initial cycle quality control as describedbelow.

Assuming that sequencing continues following the routine 104, the logicmay advance to step 106 where a routine is performed to evaluate thequality of the base addition steps of sequencing. Presently contemplateddetails of the base addition quality control routine 106 are describedbelow with reference to FIG. 7. In general, however, the base additionquality control routine will evaluate parameters of the sequencingsystem to determine whether changes should be made to the systemoperating settings or whether sequencing could or should continue underthe same or different conditions. As with the initial cycle qualitycontrol 104, the base addition quality control routine 106 may result inre-performing certain sequencing steps or even aborting the sequencingprocess altogether.

In an alternative embodiment, step 106 can be performed after step 100and prior to step 102. This order may be advantageous if the queries andsteps involved in step 106 provide information that is useful inevaluating characteristics or qualities of samples and the system atstep 104. Furthermore, as set forth in further detail below in regard toFIGS. 6-8, different queries and steps exemplified for the various QCsteps of FIG. 5 can be carried out in different orders than specificallyexemplified herein or even repeated more than once and in a variety ofcombinations to suit a particular synthetic technique or synthesissystem.

At step 108, the logic may call for determining whether the currentcycle is the last cycle of sequencing. Several scenarios may beenvisaged for this step. For example, the sequencing system may beprogrammed to perform only a predetermined number of cycles, with thesequencing terminating after the predetermined number of cycles havebeen performed. Alternatively, the quality of certain data collected bythe system may be evaluated to determine whether data of a desiredquality is still being collected. That is, as summarized below, in thequality control routines presently contemplated, a signal-to-noise ratiomay be evaluated to determine whether the base addition operation andimaging operations can adequately distinguish the type of nucleotidethat is being added at individual sites. Where such addition, or imagequality, or the ability to distinguish between the nucleotides attachingat individual sites is at an undesirable level, the system may indicatethat the current cycle is to be the last cycle for sequencing of aparticular sample array. Other sequencing ending scenarios may, ofcourse, be implemented. If the current cycle is determined to be thelast cycle, then, end run programs may be performed as indicated at step110. In general, such programs may include processing of image data,exporting of data, notifying a human operator or robot to remove thesample array container from the system, and so forth.

If the current cycle is not determined to be the last sequencing cycle,the logic may advance to step 112 where the blocking agents andfluorescent dyes are removed from the last nucleotide added at eachsite. At step 114, then, the sites or waste in the effluent stream maybe detected for additional quality control. For example, the additionalimaging of the sites or waste at step 114 may be used to determinewhether the sites were adequately de-blocked by determining whether thedyes continue to fluoresce at each site (or at control sites, asdescribed below). Alternatively, detection of the waste material maydetermine whether blocking agents that are fluorescent or that absorbradiation at a particular wavelength are present in the effluent streamat a sufficient level to indicate a desired level of de-blocking hasoccurred. Where such detection is not desired, step 114 is optional andmay be deleted from the process. At step 116, then, a quality controlroutine to evaluate the de-blocking operation is performed. Details of acurrently contemplated de-blocking quality control routine are describedbelow with reference to FIG. 8. Following step 116, the logic may returnto step 98 where additional bases are added for a subsequent cycle ofsequencing.

The logic for carrying out and controlling a sequencing operation aspresented in FIG. 5 and set forth above is merely exemplary. The logichas been exemplified in the context of particular sequencing techniques.It will be understood that the logic can be modified to accommodatedifferent sequencing techniques. For example, pyrosequencing techniquesare often carried out using nucleotides that do not have blockinggroups. Accordingly, logic for carrying out pyrosequencing need notinclude steps related to de-blocking such as steps identified as 112,114, and 116 in FIG. 5. As a further example, pyrosequencing techniquestypically utilize secondary enzymes for detection of releasedpyrophosphate, such enzymes including, for example, sulfurylase andluciferase. The logic for carrying out pyrosequencing can include addedsteps related to adding or removing secondary enzymes. Furthermore, QCsteps used in a pyrosequencing method can include steps that are relatedto querying the activity of the secondary enzymes and responding toinformation obtained from the query. Similar modification can be made tothe logic for carrying out other sequencing techniques that include useof secondary reagents, such as enzymes used for detection or nucleicacid modification. For example, cycles of sequencing-by-ligation, whichinclude a step of removing a portion of ligated probes using arestriction endonuclease or chemical cleavage of a nucleic acid strandafter detection and before initiation of a new cycle, can be covered bylogic that includes steps related to adding and removing the cleavingagent (such as the restriction endonuclease or chemical cleaving agent)or evaluating QC related to activity of the cleaving agent.

Throughout the sequencing process, a number of individual systemparameters may be monitored and regulated in a closed-loop or open-loopmanner. Again, an object of such monitoring and control is to allow forautomated or semi-automated sequencing through efficient reaction anddetection processes. In a presently contemplated embodiment, forexample, system diagnostic parameters might include temperature of thesample or the sample container, reagent temperature, temperatures atvarious locations in the instrumentation, reagent volumes and flowrates, power of light sources (particularly laser light sources), pHlevels downstream of the flow cell, humidity, vibration, presence ofozone, image intensities, focus quality, and so forth. Additionalparameters might include reagent pump pressure, the levels of reagentsremaining in reservoirs, presence of bubbles in a detection chamber(e.g., a flow cell), and computer storage space available, both forimaging data and sequence data. Moreover, in addition to these ongoingand regular checks, unusual process developments may be detected, suchas the opening of a door or other access panel at a sample insertion andretrieval station (see FIG. 2), fluid overflows, and so forth. In caseswhere the system determines that continuing the sequencing process wouldnot result in data being collected for each cycle, the system can makean automated decision to end the sequencing run or to flush reagentsthrough the flow cell to preserve the sample and enter a safe state thatpreserves the sample until data collection can be resumed. In particularembodiments, the system can indicate an error to an operator andoptionally suggest corrective measures. Alternatively or additionally,the system can make an automated diagnosis and response to the error.Thus, synthesis steps can be restarted and data collection continuedeither by operator intervention or by automated correction.

Transducers and circuitry for monitoring and controlling such parametersmay be generally similar to those available for other process systems.For example, any form of suitable temperature transducer may be used formonitoring sample, container, reagent, and instrument temperatures.Suitable flow meters may be used for monitoring reagent volumes andflows. Conventional pressure transducers may be used for detectingreagent pump pressures, back pressures, and so forth. Logic circuits forclosed-loop control or open-loop operator notification based upondetection of such parameters may include analog or digital circuits(e.g., programmed computers). In a presently contemplated embodiment,for example, the quality/process control system described above withreference to FIGS. 1 and 2 may perform these functions. As will beappreciated by those skilled in the art, the signals produced by thevarious transducers, where computer control is employed, will beconverted to digital values which can be compared to normal operatingranges, fault limits, alarm limits, and so forth. Where possible,closed-loop control may be employed to maintain temperatures, volumes,power levels, flow rates, pressures, and so forth within acceptableranges to permit continued sequencing. Where alarm or failure limits arereached, operation of the control routines preferably includesestablishing an exception or error log and storing events in the errorlog so as to permit later evaluation of the performance and operation ofthe sequencer during particular sequencing steps, over periods of time,and so forth.

Closed-loop control of such parameters may be performed to enhance thesequencing process. For example, in a presently contemplated embodiment,the fluidics control/delivery system 18 may include heaters or coolersthat can provide reagents and other fluids at desired temperatures toenhance and promote reactions with samples in the arrays. For example,heaters may be provided for elevating the temperature of the sampleduring certain portions of the sequencing process, and such temperaturesmay be regulated for the process fluids as well. Thermal transferdevices, such as heaters, coolers, heat exchangers, and so forth may beemployed for this purpose. Other closed-loop control may be performedbased upon the target parameters for the individual steps in thesequencing operation. Those skilled in the art will recognize, as well,that such parameters may be combined to determine when the sequencingsystem is operating properly, when sequencing can proceed, or when oneor more such parameters is out of a normal range to the extent thatsequencing should not proceed. In such cases, samples may be preservedat least for some duration of time until the sequencing system isoperative within its desired parameters.

FIG. 6 illustrates exemplary logic that might be included in a qualitycontrol routine for an initial cycle of sequencing. The initial cyclequality control routine 104 may be designed to examine characteristicsor qualities of samples and sample arrays to determine whetherhigh-quality and sufficient sequencing data can be obtained. In apresently contemplated embodiment, for example, the routine may beginwith a query as indicated at step 118 to determine whether too few sitesare present and detectable in the array. This step may refer to anacceptable range or number of sites that make sequencing of the arrayeconomical in terms of the amount of data that can be collected for theamount of time and materials required to process the array. The querymade at step 118 will typically be based upon the detection made at step100 summarized with reference to FIG. 5. By way of example only, in apresently contemplated embodiment, an acceptable number of sites onwhich sequencing may be performed may be about 10 million sites/cm² ormay be in a range between about 5 million sites/cm² and 100 millionsites/cm², although developing technologies will likely increase theupper end of this range in most cases to 1000 million sites/cm² orhigher. The density of sites (for example, beads) on an array can alsobe evaluated in terms of percent capacity such that an acceptable numberof sites is indicated by a capacity between, for example, 35% and 100%(100% or full capacity being based on the ideal case where sites areevenly distributed and at a distance that is just sufficient to allowadjacent sites to be distinguished). The positions of the beads or othersites may be regularly spaced with a known separation between individualsites, or a random distribution, for example, an array of amplifiedclusters or a random array of beads on a surface.

If too few sites are detected or discernible from the imaging ordetection operations performed in the initial cycle, control may bedirected to step 120 where queries may be made of the fluid delivery ordetection systems, or both systems, or other systems of the sequencer.In general, a particular density of sites or clusters will be desired.If the detected number of such sites is low or lower than desired, thismay be indicated by a count of the number of sites or by determiningthat a number of “dark” pixels (e.g., pixels not apparently indicativeof the presence of a site) is above a ceiling. Such occurrences could bedue to parameters of the fluid delivery system or the detection system,or both, as well as other parameters of the sequencer. For example,absence of detected sites may be due to inadequate delivery of labelednucleotides to the array sample or due to improper focusing of theimaging system. The query performed at step 120, then, may examineoperational parameters of the type described above to determine whetherproper operation is possible. Following such determinations, alterationsin the system settings may be performed, and the sample may be returnedfor re-focusing and re-imaging, if necessary, as indicated at step 122.Similarly, the system may be returned for re-delivery of biomoleculereagents at step 98.

It should be noted that throughout the present discussion, and indeedfor all of the quality control routines summarized in the presentdiscussion, one or more of the responses may be performed, and suchresponses may be performed in any logical order where appropriate. Forexample, for the query of the delivery and detection systems, and there-focus/re-image routine described above, these may be performed inparallel in any sequence. Moreover, for these and for other routinesperformed, the present discussion should not be considered as limiting.Depending upon the parameter data collected, the sequencing techniquebeing used and the possible cause of anomalies in operation of thesystem or in the sequencing data obtained, other routines may beperformed as well. Similarly, it may be advantageous to perform someroutines before others. For example, a quick check of the operationalparameters of the system, noting slight anomalies that have beencorrected, may be more efficient than recycling the sample in aretrograde fashion back to an imaging station as would be called for atstep 122 (assuming that the sample had been moved from the detection orimaging station). Finally, it should be noted that certain of the stepswill clearly call for repeating of certain sequencing operations,altering certain sequencing operations, or even aborting sequencing ofthe particular sample as denoted by the arrows extending to the right inFIGS. 6, 7, and 8. It may be considered that following the variousresponse routines described herein, a determination is made as towhether the condition that led to the action has been remedied, suchthat sequencing may proceed albeit by the return of the sample to apreceding operation.

In addition to determining whether too few sites are present in thesample, the initial cycle quality control routine, examining qualitiesof the sample, may determine whether too many sites or an uneven sitedistribution is present, as indicated generally at reference numeral124. Because certain sample preparation techniques may result in anoverabundance of sites, or sites that may too closely approach oneanother, there may be a desired limit to the number of sites in aparticular sample, or to the relative density or congestion of sites inone or more regions of the array. Other indicators of sample qualitythat can be queried at this step include the size, shape, or morphologyof sites. Typically, sites will have an expected size, shape, ormorphology and deviations can be indicative of a particular problem. Forexample, if sites are too densely packed then a large fraction of siteswill overlap each other such that overlapped sites appear as a singlesite having an apparent size that is larger than the size expected for asingle discrete site. Similarly, sites that overlap can be identified byan apparent shape that is different than expected for a single site,such as in the case of typically circular sites that will appear as asingle hourglass shape when two sites overlap. Other aberrations insize, shape, or morphology of sites can be indicative of problems inpreparation of the array that occurred prior to loading the array in thesystem such as insufficient amplification at one or more sites orexcessive amplification at one or more sites. If, upon the evaluation ofthe data collected at step 100 in FIG. 5, it is determined that too manysites or an undesirable site distribution is present, several approachesmay be envisaged in response. At step 126, for example, the fluiddelivery and detection systems may be again queried in a manner similarto that discussed with reference to step 120 above. Moreover, theimaging system may be re-focused, particularly if the detection dataindicates that inadequate or unreliable image data was obtained that mayhave led to the determination at step 124. The re-focusing andre-imaging step 128 may be essentially similar to that performed at step122 above, and may require return of the sample to the imaging stationif it has been moved from the imaging station.

Another response to the presence of too many sites or an uneven sitedistribution could be the masking of certain regions or sites andignoring image data from such regions during processing. The maskingresponse, indicated at step 130, would generally include development ofa digital mask for the pixilated images in which particular locationscorresponding to particular sites are designated by a first value, andsites to be analyzed are designated by a second value. Such a binarymask would generally be stored as a lookup table that permits comparisonof the location of mask pixels in subsequent sequencing cycles so thatdata for such locations would not be processed for analysis andsequencing. It is possible, however, that such masking could result inelimination of too many sites or even large regions of the sample arrayfrom sequencing such that pursuing further processing of the sample isnot economical or is otherwise undesirable. At step 132, then, it may bedesirable to determine whether the masking has resulted in too fewsites. As with step 118, this inquiry may essentially consist ofdetermining whether the number of remaining sites after digital maskingmake sequencing worthwhile in terms of the amount of data that can becollected. If too few sites are available for sequencing after themasking of step 130, the mask may be re-evaluated as indicated at step134, such as to determine whether certain sites can be reliablysequenced. The mask may then be altered accordingly and sequencing mayproceed. If too few sites are available for sequencing, sequencing maybe interrupted altogether.

The amount of image data processed during sequencing operations tends tobe massive and even quite overwhelming at times. As such, analysis ofthe data can prove somewhat onerous unless the data is collected,organized, and managed efficiently. Therefore, it may be advantageous toprocess the data such that useful data is saved and prepared for furtherprocessing while discarding data which has a high probability of notbeing useful. Accordingly, the use of masking, as well as other imageprocessing utilities, may be coordinated to attain the overall goal ofworthwhile sequencing data. With this in mind, it should be noted thatthe image data collected may be processed in various ways. For example,areas on a test sample may be selected to be imaged while other areasmay be selected to be bypassed. If this type of selective imaging isdone, several different options may be used to handle the data. In oneembodiment, image data for only the areas flagged as areas of interestmay be collected while image data for other areas may not be collected,or may be collected but not retained or not analyzed. In an alternativeembodiment, image data may be collected for all areas of the sample butthe data may be stored in different locations. For instance, the imagedata flagged as areas of interest may be stored in a first databasewhich is used for sequencing analysis whereas the other image data maybe stored in a second database. In addition, logging of image datacollection may follow similar procedures, such as only logging activityfor certain imaging or logging activity for all imaging but saving thelogs in various locations. Also, the selective processing of image datamay be based on any parameter collected during sequencing operationsincluding, but not limited to, chemistry parameters, environmentalparameters, and so forth. Therefore, in general, the image data may behandled using various selective processing schemes based on variousprocesses including, but not limited to, the masking methods discussedabove.

If any of the responses indicated at steps 126, 128, 130, or 134, or theresponse to the query 132 enable sequencing to proceed, then, theinitial cycle quality control routine may be exited and sequencing maycontinue as summarized above with reference to FIG. 5.

FIG. 7 illustrates exemplary logic for performing a base additionquality control routine 106 as described generally above with respect toFIG. 5. The routine may be performed at various stages in sequencing butwill likely be performed after imaging of the array in each sequencingcycle but before image data is used to determine sequence data. Theroutine is essentially designed to determine whether the detectionprocess proceeded as desired, or whether sequencer parameters should beadjusted to provide for improved imaging and detection. Because thequality of the detection performed on the sample will ultimately affectthe quality of the sequencing data, it may be desirable thathigh-quality images be returned and it may be most useful to performsuch base addition quality control routines for each and everysequencing cycle. Moreover, as discussed above, the routine may be atleast one of the considerations in determining whether sequencing shouldcontinue or whether the present or even the previous sequencing cycleshould be considered the last reliable cycle in which sequencing datashould be retained or evaluated.

In the embodiment illustrated in FIG. 7, an initial query 136 determineswhether the general image quality is acceptable. For example, while asharp image, and particularly a consistently sharp image over the lengthand width of the array is desired, such factors as poor focus may resultin an unacceptably blurred image. The evaluation of image quality maytake a number of forms. In a presently contemplated embodiment, forexample, a focus score is attributed to each image. The focus score maybe based upon sharpness of the image, sharpness of particular featuresor marks in the image, anticipated structures visible in the image,gradients of intensities or colors detectable in the image, and soforth. Image quality can be based on an image of all or part of thearray. An advantage of evaluating only a part of the array is that animage can be obtained more rapidly for purposes of determining qualityprior to expending the time on obtaining a full image. If the imagequality is found to be unacceptable, steps 138 and 140, or othersuitable steps, may be performed in response. Steps 138 and 140 maygenerally correspond to steps 120 and 122 described above with referenceto FIG. 6. That is, the fluid delivery and/or detection systems may beevaluated to determine whether their operating parameters are within theacceptable ranges, or the imaging system may be re-focused and thesample returned for imaging if it has been displaced from the imagingstation.

Another aspect of image quality that may be monitored is the presence ofbubbles within the sample. If bubbles are detected, the image data, orportions of the data, may not be adequate for further processing. Forinstance, the image data may appear to have blurry regions or regionswhere detected colors are indistinguishable. Furthermore, the presenceof bubbles may signify an underlying problem with a particular sample.For instance, the bubbles may be impeding the nucleotides from attachingto the sample. The presence of bubbles may be monitored within the fluidchannel via a photodiode or other detector such as one that isconfigured to monitor changes in the signal received at the dioderesponsive to the refractive index differences between air and liquid.If bubbles are detected, any number of suitable response steps may beperformed. For instance, the situation may warrant returning the sampleto a particular fluidics station and performing the base addition stepagain. In cases where the channels are filled from top-bottom with airrather than liquid, it is possible to automatically adjust the focaldepth to restore the focus of the “dry” image, or simply to flow moreliquid through the channel in order to remove the air bubble. Anotherpossible response may include masking of certain regions or sites andignoring image data for regions or sites determined to contain bubbles.Yet another response may include interrupting sequencing operations onthe sample if it is determined that the bubbles are such a detrimentthat proper imaging is no longer possible.

It should be noted that a query similar to that of step 136, andresponses such as those summarized at steps 138 and 140 may also be partof the initial cycle quality control routine summarized above withreference to FIG. 6. That is, it may be possible that the control ofquality of the samples is compromised by poor functioning of the fluiddelivery system or the detection system. In such cases, routines such asthose intended at steps 138 and 140 may also be performed to ensure thatthe sample quality evaluation proceeds on the basis of reliableinformation. Where desired, the parameters of the sequencing system maybe adjusted and the sample may be re-imaged, and the sample qualityre-evaluated based upon improved input data.

As noted above, the sample arrays may be designed to facilitate certaintypes of quality control. For example, control clusters or sites may beincluded in the array that have known sequences of nucleotides. Suchknown sequences may, for example, be repeating sequences of the fourcommon DNA nucleotides. Alternatively, such control sites may includehomopolymer sequences of a single nucleotide type. The quality controlperformed in the routine 106 may rely upon expected results for suchcontrols during successive sequencing and imaging steps carried out inparallel with sites of unknown composition for which sequenceinformation is desired. As indicated at step 142, then, evaluation ofsuch control sites may be made to determine whether the anticipatedaddition of a base has been detected. Because the sequence of suchcontrol sites is known, such evaluation may determine, for example, thatno base was added, the wrong base was added, or a low yield for basecoupling was detected (e.g., an anticipated characteristic dye color ata control site was weak in intensity or was obscured by another color).Another type of control that can be included is a site having a labelmoiety directly attached. For example, in embodiments directed tosequencing using fluorescent labels, a site can include the fluorescentlabels directly attached (i.e. not via a hybridized oligonucleotide) toserve as a control for detection quality that is independent of otheraspects of the sequencing chemistry (such as efficiency of hybridizationand nucleotide addition).

The failure to add a base may be indicated by a single intensity in theimage data that is below a desired threshold. The addition of a wrongbase may be indicated by a different color signal being detected in theimage data (e.g., at a control site) than was anticipated. An indicatorfor a low yield base coupling may, as indicated above, be a signalintensity that is lower than expected, similar to the test for no basehaving been added. The expected intensity can be a particular thresholdlevel that remains unchanged for all cycles. Alternatively, thethreshold level can be reduced at each cycle in accordance with anacceptable loss of yield at each step or in accordance with anempirically determined loss of yield determined from the signal detectedfrom one or more previous cycles, as described for example in regard tosignal-to-noise (S/N) ratio below.

Several responses may be envisaged for improving sequencing, image data,or sequence data where query 142 determines that the imaging of controlsites was defective. For example, as indicated at step 144, the systemmay query the fluid delivery and detection systems to determine whetheroperating parameters are within acceptable ranges. Alternatively, or inaddition to this step, the base addition process may be repeated asindicated at step 146. In general, step 146, as with the re-imagingsteps described above, may require that the sample be returned to afluidics station for addition of the bases.

In addition, several parameters may be used to help monitor imaging andsequencing operations. As mentioned in various passages throughout thisdisclosure, these may include parameters relating to chemistry (e.g.,evaluating reagent delivery), parameters relating to fiducials (e.g.,control clusters), sample site parameters (e.g., site quality,distribution, shape, number, and so forth), and temperature parameters(e.g., fluid temperature, array temperature, instrument temperature, andso forth). However, many other parameters may prove useful inascertaining how successfully the sequencing operations are proceeding.For instance, various environmental parameters may be monitored toprovide input as to how external factors may be affecting sequencingoperations. These environmental parameters may include, withoutlimitation, humidity, external power sources, temperature, vibration,and so forth. In addition, it may prove useful to monitor pH levelsdownstream of the flow cell. Doing so may yield insight as to howeffectively the steps of base addition, blocking, de-blocking, andwashing are progressing. It may also be desirable to monitor any phasingoccurring between the individual sample sites. For instance, individualcopies of a sequence at a sample site may experience cycles wherenucleotides do not attach. The result is a site having heterogeneity inthe length of the extended species. If at each cycle the number oftruncated copies increases, then eventually the fraction of copies atthe site that have been extended at every cycle is reduced. This resultsin the site having copies that are out of phase and a perceivedreduction in S/N ratio. Eventually, this may lead to a situation wherethe S/N ratio degrades to such a level that sequencing data becomesunreliable. Early detection of flow cells that show high levels of siteshaving phasing problems can allow measures to be taken to ensure thesample is not completely lost, for example changing the sequencingreagents or checking the fluidics of the instrument. Alternatively oradditionally, a decision can be made to halt reagent delivery to asample having an undesirable number of out of phase sites. This canprovide the advantage of reducing sample waste. Tracking prematurephasing problems can provide the basis for a determination of thefunctionality of the instrument which can be responded to by alterationsmade by an operator or in an automated fashion according to predictedcauses.

A further query that may be made in the base addition quality controlroutine is indicated at step 148, and may consist of determining whetherthe S/N ratio is within an acceptable limit. In general, as noted above,detection of colors of fluorescent dyes for individual sites may be abasis for determination of sequence data, and the ability to accuratelydetect such colors may be important for obtaining reliable sequencedata. A poor S/N ratio may be determined, for example, by comparingintensities or colors for individual sites, or for control sites, to S/Nratios for similar sites in previous cycles. It may be anticipated that,due to a statistically acceptable decay in yield for nucleotide couplingover a series of sequencing cycles, a normal decay in S/N ratio shouldbe anticipated. Indeed, the determination of whether sequencing shouldproceed through an additional cycle or even whether sequence data orimage data should be analyzed or stored for a current cycle may bedetermined by reference to the decay in the S/N ratio or in an objectivelimit of this ratio. Where the S/N ratio decays abnormally or in acatastrophic manner, several responses may be in order, for example achange of reagents such as the scanning buffer on the instrument beforeundertaking a further cycle of sequencing chemistry and detection. Itshould be noted that as an alternative, or in addition to analyzing thedecay in the S/N ratio, a decay in a signal from de-blocking agents inan effluent stream may be made.

Responses to a decrease in S/N ratio detected at step 148, in additionto termination of sequencing, may include querying the fluid deliveryand detection systems as indicated at step 150, such as to determinewhether the systems are operating within their normal parameters, orshould be adjusted to permit further sequencing. Alternatively, or inaddition to this, the sample may be re-imaged with a higher exposurelevel (e.g., higher power output for the light sources), a highersensitivity in the detection algorithms, or a change in any otherparameter that might permit the S/N ratio to be improved (step 152). Forexample, the time of exposure may be lengthened to allow for morephotons to be collected in particular images. It may also be desirableto alter imaging parameters such as the scan rate to allow for sites tobe more accurately detected, or in higher resolution. Other parametersthat can be changed are the conditions for nucleotide addition. Forexample, as the S/N ratio reduces, each subsequent cycle can be carriedout with longer incubation times or increased concentration of reagentsto help better drive the nucleotide addition reaction to completion. Ifthe S/N ratio can be improved in such manners to an acceptable level,sequencing may continue.

FIG. 8 illustrates presently contemplated logic for a de-block qualitycontrol routine 116 as discussed generally above with reference to FIG.5. In general, this routine is designed to determine whether dyes andde-blocking agents have been adequately removed from the last addednucleotides at the individual sites such that another nucleotide can beadded in a subsequent sequencing cycle, and to ensure that the dyes ofpreviously added nucleotides will not interfere with imaging ofsubsequently added nucleotides. As before, several queries may be inorder to determine whether de-blocking has been adequately performed. Atstep 154, for example, a query may be made as to whether an image madeafter de-blocking is adequate for analysis. As discussed above withreference to step 136 of FIG. 7, several factors may causeinsufficiently sharp or detailed images to be obtained. In a mannersimilar to response 138 and 140 of FIG. 7, then, the fluid delivery anddetection systems may be queried, and the imaging system used to imagethe sample after de-blocking may be re-focused and sample may bere-imaged, as indicated at steps 156 and 158, respectively. As will beappreciated by those skilled in the art, the fluid delivery systemexamined at step 156, however, will be evaluated to determine whetherreagents for cleaving de-blocking agents and fluorescent dyes isoperating within normal limits or in a desired manner.

If the responses at steps 156 and 158 can adequately remedy thecondition, a subsequent query 160 may be made. Step 160 is essentiallysimilar to step 148 summarized above with reference to FIG. 7. That is,the system may determine whether a S/N ratio is sufficiently high topermit proper analysis of de-blocking. If the ratio is not sufficientlyhigh or is not within an acceptable range, responses may include againquerying the fluid delivery and detection systems, and re-imaging thede-blocked sample with modified imaging settings, as indicated at steps162 and 164, respectively.

A further query in the exemplary routine 116 may include determiningwhether de-blocking agents have been sufficiently removed, as indicatedat step 166. Two tests are presently contemplated for such evaluation,which may be performed in the alternative or both tests may beperformed. In general, a first test may be based upon evaluation ofcontrol sites of the type discussed above. Such sites may be imaged todetermine whether the anticipated color change (e.g., essentially thedisappearance of the site from the image) has occurred. If the controlsites do not indicate that effective de-blocking was performed, thede-blocking operation may be repeated as indicated at step 168. Again,repeat of the de-blocking operation may require return of the sample toa de-blocking station. If desired, sites other than control sites canalso be imaged so as to query whether or not de-blocking has occurred.

Another test for de-blocking may be the evaluation of de-blocking agentsin waste or an effluent stream following the de-blocking step. As willbe appreciated by those skilled in the art, blocking agents may becoupled to dyes that become active and can fluoresce once the blockingagent has been removed from the nucleotides. The blocking agent can alsobe detectable in the effluent by absorbance at a particular wavelength.The waste stream may be tested, for example using an inline detectordirected to the effluent stream, to determine whether sufficientblocking agent is detected in the waste stream. If insufficient blockingagent is detected, the possible responses may be to query theperformance parameters of the fluid delivery system of the processfluids used for the de-blocking reaction and/or to repeat thede-blocking operation, as indicated at steps 170 and 172, respectively.Depending upon the sequencing chemistry used, a single moiety on theadded nucleotides may serve as both blocking group for preventingextension and as a label for detecting nucleotide addition or,alternatively, added nucleotides can have separate label and blockingmoieties. The methods set forth herein with regard to determiningremoval of a blocking group are intended to be illustrative of methodsfor determining removal of a label moiety and/or blocking moiety eitherseparately or together. For example, in embodiments using separate labeland blocking moieties, the moieties can be removed and detectedseparately or together in the effluent using methods similar to thoseexemplified above with regard to detecting a blocking group.

It should be noted that in all of the steps summarized in FIGS. 6, 7,and 8, logs of the operations performed and any remedial measures taken,are preferably kept. The logs may also be associated with the individualsamples, and may be time-stamped to evaluate proper performance of thesequencing system. Where the sequencing operation is attended or can beattended by one or more operators, notification by visual or audiblealarms may be provided to the operator indicating that attention to oneor more samples or attention to one or more stations in the sequencingsystem may be in order.

It should also be noted that a substantial temporal decoupling of thesequencing steps and remedial measures taken in the quality controlroutines may exist in accordance with the present invention. That is,while sequencing systems may be established to process multiple samplesand sample containers, these need not be processed through the system inany particular order, or even at the same rate. Based upon the qualityof the sample, process parameters, and the outcome of the variousquality control steps and routines, for example, certain samples mayundergo some degree of regressive flow through the sequencing stationsand sequencing steps. In certain cases, samples may be set aside or leftout of the system for certain periods of time for evaluation of eitherthe sample or the system, or both. The system control circuitry ispreferably designed to track individual samples and the sequencingperformed regardless of whether samples are taken out of sequence, takenin various times, or even whether samples require longer or shortertimes for the various reactions, imaging, evaluation, and so forth. Suchtemporal decoupling may be an important feature in promoting efficientoperation and high throughput of automated or semi-automated parallelsequencing of samples.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

1. (canceled)
 2. A method for determining nucleotide sequences for aplurality of nucleic acids, comprising: (a) performing a cycle of asequencing procedure for an array comprising a plurality of nucleicacids, wherein the cycle comprises directing radiation from a laserlight source to the array at an exposure level and detectingfluorescence emission from the array at the exposure level; (b)increasing the exposure level of the radiation directed to the array toa second exposure level that is higher than the exposure level; (c)performing a cycle of the sequencing procedure by directing radiationfrom the laser light source to the array at the second exposure leveland detecting fluorescence emission from the array at the secondexposure level; (d) increasing the exposure level of the radiationdirected to the array to a third exposure level that is higher than thesecond level; and (e) performing a cycle of the sequencing procedure bydirecting radiation from the laser light source to the array at thethird exposure level and detecting fluorescence emission from the arrayat the third exposure level, thereby determining nucleotide sequencesfor the plurality of nucleic acids.
 3. The method of claim 2, whereinthe exposure level is increased by increasing the power output for thelaser light source.
 4. The method of claim 2, wherein the exposure levelis increased by increasing the time of exposure of the array to theradiation.
 5. The method of claim 2, wherein the plurality of nucleicacids are attached to individual sites of the array.
 6. The method ofclaim 2, wherein each cycle of the sequencing procedure comprises addingnucleotides to the plurality of nucleic acids of the array.
 7. Themethod of claim 2, wherein each cycle of the sequencing procedurecomprises adding oligonucleotides to the plurality of nucleic acids ofthe array.
 8. The method of claim 2, wherein each cycle of thesequencing procedure comprises obtaining image data from thefluorescence emission.
 9. The method of claim 8, further comprisingprocessing the image data to identify individual sites of the array. 10.The method of claim 2, wherein each cycle of the sequencing procedurecomprises delivering to the array a fluid comprising sequencing reagentsand then removing the fluid from the array.
 11. The method of claim 10,wherein each cycle of the sequencing procedure further comprisesdelivering to the array a wash fluid to remove sequencing reagents fromthe array.
 12. The method of claim 2, wherein twenty five or moresequencing cycles are performed.
 13. The method of claim 2, wherein thesteps are carried out automatically.
 14. The method of claim 2, whereinthe steps are carried out with input from a human operator.
 15. Themethod of claim 14, wherein a system control interface generates arecommendation to the human operator regarding one or more of the steps.16. The method of claim 2, further comprising a step of evaluating adecay in signal-to-noise ratio of the fluorescence emission from thearray.
 17. The method of claim 16, wherein the exposure level isincreased based upon the decay in the signal-to-noise ratio.